Objective ·To compare pregnancy outcomes between broad-spectrum antibiotics administered after hysteroscopy and 16S rRNA gene sequencing combined with targeted antibiotics and Lactobacillus supplementation in patients with recurrent implantation failure (RIF) and chronic endometritis (CE). Methods ·This retrospective study analyzed 676 RIF patients who underwent either a 16S rRNA test during the mid-secretory phase or hysteroscopy during the proliferative phase at the Reproductive Medicine Center, Department of Obstetrics and Gynecology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, from January 2023 to May 2025, after excluding known factors (preimplantation genetic testing for aneuploidies was not performed). Twenty-seven patients with 16S rRNA results indicating Lactobacillus abundance <90% and single-pathogen abundance≥10% were included in the 16S rRNA-diagnosed CE group (16S rRNA group). These patients received targeted antibiotics based on the pathogens for 7‒10 d, followed by vaginal Lactobacillus supplementation for 10 d. Sixty-five patients were diagnosed through hysteroscopic examination and CD138 immunohistochemical testing and were included in the hysteroscopy-diagnosed CE group (hysteroscopy group). They received levofloxacin combined with metronidazole for 14 d. All patients commenced frozen embryo transfer (FET) cycles in the subsequent menstrual cycle after treatment. Propensity score matching (1:4) was performed on FET cycles from the two groups using 13 covariates affecting pregnancy outcomes. The primary endpoint was the live birth rate. The secondary endpoints included the clinical pregnancy rate, human chorionic gonadotropin (HCG)-positive rate, miscarriage rate, and the incidences of pregnancy complications and neonatal adverse outcomes. Multivariate Logistic regression analysis was performed to assess potential risk factors affecting live birth. Results ·In the hysteroscopy group, 65 patients underwent 127 FET cycles, while in the 16S rRNA group, 27 patients underwent 33 FET cycles. Among them, 47 patients (74 FET cycles) from the hysteroscopy group and 22 patients (26 FET cycles) from the 16S rRNA group were successfully matched. After matching, the 16S rRNA group had significantly higher live birth rate (61.5% vs 23.0%, P=0.001), clinical pregnancy rate (65.4% vs 33.8%, P=0.011), and HCG-positive rate (73.1% vs 46.7%, P=0.038) than the hysteroscopy group. There were no significant differences in miscarriage rate, ectopic pregnancy rate, or the incidences of pregnancy complications and neonatal adverse outcomes between the two groups (all P>0.05). Multivariate Logistic regression analysis showed that the hysteroscopy group was associated with a lower live birth rate (OR=0.230, 95%CI 0.087‒0.607, P=0.003). Additionally, advanced maternal age, increased body mass index (BMI), transfer of only one embryo, and transfer of cleavage-stage embryos were all associated with a lower live birth rate (all P<0.05). Conclusion ·Compared with broad-spectrum antibiotics following hysteroscopy, 16S rRNA sequencing-based microbiota testing combined with targeted antibiotic therapy and Lactobacillus supplementation more effectively improves pregnancy outcomes in RIF patients with CE, and no adverse effects on maternal and fetal safety have been found.